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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy ARTICLE IN PRESS Ultramicroscopy 109 (2009) 344–349 Contents lists available at ScienceDirect Ultramicroscopy journal homepage: www.elsevier.com/locate/ultramic A fluorescence scanning electron microscope Takaaki Kanemaru a, Kazuho Hirata b,Ã, Shin-ichi Takasu c, Shin-ichiro Isobe d, Keiji Mizuki e, Shuntaro Mataka f, Kei-ichiro Nakamura g a Morphology and Core Unit, Kyushu University Hospital, Kyushu, Japan b Department of Anatomy and Cell Biology, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Maidashi 3-1-1, Fukuoka 812-8582, Japan c Advanced Technology Division, JEOL Ltd., Tokyo, Japan d Department of Applied Chemistry and Biochemistry, Faculty of Engineering, Kyushu Sangyo University, Fukuoka, Japan e Department of Nanoscience, Faculty of Engineering, Sojo University, Kumamoto, Japan f International Science Technology Co., Ltd, Kasuga Laboratory, Fukuoka, Japan g Department of Anatomy, Kurume University School of Medicine, Kurume, Japan article info abstract Article history: Fluorescence techniques are widely used in biological research to examine molecular localization, while Received 12 August 2008 electron microscopy can provide unique ultrastructural information. To date, correlative images from Received in revised form both fluorescence and electron microscopy have been obtained separately using two different 22 December 2008 instruments, i.e. a fluorescence microscope (FM) and an electron microscope (EM). In the current Accepted 6 January 2009 study, a scanning electron microscope (SEM) (JEOL JXA8600 M) was combined with a fluorescence digital camera microscope unit and this hybrid instrument was named a fluorescence SEM (FL-SEM). In Keywords: the labeling of FL-SEM samples, both Fluolid, which is an organic EL dye, and Alexa Fluor, were Correlative microscopy employed. We successfully demonstrated that the FL-SEM is a simple and practical tool for correlative Scanning electron microscopy fluorescence and electron microscopy. Fluorescence microscopy & 2009 Elsevier B.V. All rights reserved. Organic EL fluorophore 1. Introduction localization. Fluorescent probes, which label an identical region of a single specimen not only for fluorescence microscopy but also Fluorescence microscopy has become an indispensable micro- for electron microscopy [6], such as FluoroNanogold [7], and more scopy technique for the examination of biological specimens, recently ReAsH, which is used in the tetracystein–biarsenical because it allows selective and specific detection of molecules at system [8] and small nanocrystals (Quantum dots; QDs) [9], have small concentrations with a good signal-to-background ratio [1,2]. all been being utilized; nevertheless, complications during the It even allows one to work with intact samples, including living specimen preparation process and during imaging with both types cells, and to see samples with the naked eye; these advantages are of microscopy are unavoidable. not available with other methods, such as electron microscopy [3]. One of the ways in which the complex methods for correlative Furthermore, recent developments in fluorescence imaging microscopy could be improved would be to incorporate one techniques have enabled the well-known Abbe barrier of about microscope into another. The technology of the scanning electron 200 nm lateral resolution to be crossed, that is, the diffraction microscope (SEM) is now well advanced and the image resolution limit for an optical microscope has approached the level of 100 nm of the SEM approaches that of the transmission electron as with 3D structural illumination microscopy (3D-SIM) [4] microscope (TEM) [10]. It is clear that for the majority of biological or even less than 100 nm, as with other techniques such as 4Pi, samples, it is easier and less time-consuming to prepare samples simulated emission depletion (STED) and photoactivated localiza- for scanning electron microscopy than for transmission electron tion (PALM) [3,5]. microscopy. Furthermore, the structure of the SEM makes it easier In order to obtain a stable image with a higher resolution, and more cost-effective to incorporate other devices into it. This however, an electron microscope is still required. Corre- led us to attempt to make a hybrid ‘‘FL-SEM’’ instrument in which lative study using both fluorescence and electron microscopy is an SEM is combined with an FM. In parallel with its set-up, some usually employed to obtain substantial information on molecular fluorophores were tested for the labeling of biological samples using the new instrument. We found that an organic EL fluorophore named Fluolid (Pub. no. US 7015002) [11], which has a high physical stability was suitable for this purpose. à Corresponding author. Tel.: +8192 642 6048; fax: +8192 642 6050. The design of the FL-SEM is described and the first FL-SEM E-mail address: [email protected] (K. Hirata). images are presented. 0304-3991/$ - see front matter & 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ultramic.2009.01.002 Author's personal copy ARTICLE IN PRESS T. Kanemaru et al. / Ultramicroscopy 109 (2009) 344–349 345 Some of our findings have been previously reported in abstract possible fluorescence damage which may be caused by the form [12]. electron beam. Each of the FM and SEM images is separately displayed on a single PC screen through the CCD camera and the AD converter, respectively (Fig. 1C). Both images are then 2. Experimental manually merged using Adobe Photoshop CS. 2.1. Assembly of the FL-SEM 2.2. Animals An SEM for wavelength dispersive spectrometry (WDS) (JEOL Under deep anesthesia induced by diethyl ether, adult male JXA8600 M, JEOL, Tokyo Japan) was reconstructed to create a Wistar rats were perfused intracardially with PBS, followed dual-mode microscope, named the FL-SEM, which is capable of by a mixture of 2.8% paraformaldehyde, 0.2% picric acid and obtaining both FM and SEM images without requiring to move the 0.06% glutaraldehyde in 0.1 M phosphate buffer (PB) at pH 7.4. specimen (Fig. 1A and B). All the parts of the built-in optical The diaphragm and the kidney were removed and postfixed with microscope unit in the SEM for WDS, with the exception of a 4% paraformaldehyde in 0.1 M PB. mirror and the Cassegrain type (non-chromatic aberration type) These experiments were reviewed by the Committee on Ethics 45 , NA 0.41 optical objective lens, both of which are placed  for Animal Experiments of the Faculty of Medicine, Kyushu within the column and have a small hole for the electron beam to University and were carried out according to the guidelines for pass through, were replaced with an FM fitted with a digital Animal Experiments of the University, and Law no. 105 and camera unit that was assembled in our laboratory. The unit Notification no. 6 of the Japanese Government. consisted of a laser light source from an external unit (473 nm, Showa Optronics, Tokyo, Japan) (a in Fig. 1A and B), an emission filter (515 nm LP), an adaptor device for concentrating the laser 2.3. Exploitation of a new probe for FL-SEM light (b in Fig. 1A and B), a unit consisting of mirrors and prisms (c in Fig. 1A and B), an external CCD camera (Bitran Corporation, In a preliminary study, we attempted to use some tissues Saitama, Japan) adapted via a C-mount adaptor (d in Fig. 1A and that had been labeled with GFP or FITC as FL-SEM samples. B), and an eye piece (e in Fig. 1A and B). In the FL-SEM, an electron These fluorophores were almost bleached out in the process of beam passes through small holes at the center of a mirror and the dehydration. For this reason, we employed a new probe, Fluolid, object lens in order to reach the specimen. The SEM image is then which is a small organic fluorophore originally synthesized as an sent to an AD converter (SemAfor, JEOL SA20) via a photomulti- organic EL dye in our laboratory (Pub. no. US 7015002), [11]. The plier (PMT). On the other hand, the excitation beam from the Fluolid dye has a high physical stability and a large Stokes shift external unit reaches the specimen along the same passage as the and it shows strong fluorescence intensity in its solid state. electron beam within the column. The fluorescence emission from However, it has never been applied to the labeling of biological specimens is then directed to an external CCD camera. In practice, samples. In order to test its viability, a kidney was immersed in the digital image from the FM is acquired first, with the image 20% sucrose in PBS and 10-mm-thick frozen sections of the kidney from the SEM being sequentially acquired, in consideration of were made for peanut agglutinin (PNA) staining, in accordance Fig. 1. (A). A view of the FL-SEM. (B). A schematic diagram of an inside view of the FL-SEM, which is made up of a combination of the SEM and the FM units. (C). A schematic diagram depicting the flow of SEM and FM image signals to a PC display. (a): the laser light source of the external unit (473 nm), (b): an adaptor device for laser light, (c): mirror and prism, (d): external CCD camera, and (e): eye piece.